This proposal focuses on the cloning and study of the two arginase genes in man, first as a means of elucidating their function, regulation and evolution, and ultimately as a means of devising approaches to replacing the deficient arginase (AI) activity in patients with hyperargininemia and progressive neurological and intellectual deterioration. These studies will complete the cloning of the cDNA for the mitochondrially-located form of arginase (AII) and the isolation and characterization of the genes for the cytosolic ("liver") form (AI) and AII. Clones derived from these studies will be used to define the cell biology and regulation of these genes and their protein products. They will also be used as reagents in a series of gene transfer studies designed to elucidate the tissue- and function-specific control of the AI and AII gene loci. These studies will utilize AI and AII cDNAs under the control of heterologous promotors, as well as constructs of the native gene and regulatory sequences, and reporter genes. Through the molecular manipulation of signal sequences, we will explore the importance of subcellular compartmentalization in the physiologic function of AI and AII and as a novel approach to gene therapy by redirecting the transferred gene products into or out of the mitochondrion. These studies will define the strategy and tissue specificity for the use of the gene for AI, AII or a combination of the two for gene therapy, or for the recruitment of AII as a means of "autoenzyme replacement". Finally, this project will continue to exploit the only known natural regulatory mutation in mammals, that for the expression of AI in the red blood cell of Macaca fascicularis. The use of this model is likely to provide fundamental insights into the evolution of tissue specific gene expression in higher organisms.